Nutrient agar plate

Silver nanoparticle producing bacteria was isolated from soil sample that was collected from the Annamalai University campus, Tamilnadu, India, using serial dilution technique and 0.1 ml sample was spreaded on Nutrient Agar plates (Hi Media Laboratories Ltd., Mumbai, India) and incubated at 37 ^°C for 24 h. After incubation, the plates were checked for white, swarmed mucoid colonies and then biochemical characterisation was performed to tentatively identify the isolate as Bacillus sp. The culture was finally subjected to strain identification and was confirmed using 16SrRNA primers: 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492 R (5′- TAC GGT TAC CTT GTT ACG ACT T-3′). The gene sequence obtained from the organism was compared with other Bacillus strains for pairwise identification using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and multiple sequence alignments of the sequences were performed using Clustal Omega version of EBI (www.ebi.ac.uk/Tools/msa/clustalo). Finally Phylogenetic tree was constructed by Clustal Omega of EBI (www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny) using neighbor joining method.

The MRSA and STEC stock was collected as a courtesy from the Department of Microbiology and Hygiene, BAU, Mymensingh. A loop-full of MRSA inoculum was streaked onto Mannitol salt agar (Himedia, India) plate and 5 μl of STEC stock was inoculated into the nutrient broth for enrichment. After enrichment, the broth culture was streaked onto nutrient agar plates (Himedia, India) and aerobically incubated 37°C overnight at to obtain a pure culture. The preparation of all the agar and broth was done in accordance with the manufacturer’s instruction (Himedia, India).

All MSA plates were incubated for 24 hours at 37°C. After incubation, isolated colonies suspected to be Staphylococcus were allowed to grow on nutrient agar plates (HiMedia, India) and then identified microscopically, biochemically, and serologically [19 (link)20 (link)]. For microscopic observation, a pure colony was selected and subjected to Gram staining. Then the shape, arrangement, and Gram reactions of the isolates were observed under a light microscope (Max-plank-Ring 21 D-65205, Wiesbaden, Germany) (at a magnification of 100x) [25 ]. Required confirmatory biochemical tests including catalase and triple sugar iron agar tests were performed to identify suspected S. aureus following standard protocols [25 ].